The reactions for hydrolysis or base exchange of phospholipid are generally carried out in a suitable solvent in the presence of enzyme. These reactions are usually stopped by deactivating the enzyme with a treatment such as heating. For example, Japanese Laid-Open Patent Publication No. 2-273536 discloses a method for manufacturing lysophospholipids by partially hydrolyzing derivatives of phosphatidic acid with a treatment by lipase or phospholipase A2 (hereinafter referred to as “PLA2”). In this method, the reaction mixture is heated at an appropriate point to deactivate the enzyme and halt the reaction. Japanese Laid-Open Patent Publication No. 2001-186898 discloses a method for manufacturing a phosphatidylserine by achieving a phosphatidyl group exchange reaction between an acylglycerophospholipid and serine by using phospholipase D (hereinafter referred to as PLD). In this method, the enzyme is deactivated with a treatment such as heating or alcohol denaturation after the reaction. Japanese Laid-Open Patent Publication No. 2003-319793 discloses an acylglycerophospholipid phosphatidyl group exchange reaction by using PLD. The exchange reaction is followed by deactivating the PLD with a treatment such as heating.
As described in Japanese Laid-Open Patent Publication No. 63-233750, however, phospholipases have strong thermal resistance and thus are not sufficiently deactivated even after heating, for example, at 95° C. for about 30 minutes. The inadequate deactivation of phospholipases may cause quality problems, such as odors occurred in the hydrolysis of fats in the phospholipids. This document further teaches that deactivating the phospholipases by heating at a temperature of 100° C. or more, such as 120° C., is problematic in that phospholipids or free fatty acids resulting from treating the phospholipids with phospholipases are prone to deteriorate. Japanese Laid-Open Patent Publication No. 63-233750 discloses processing a starting material that contains phospholipid with a phospholipase, then treating the phospholipase with a protease, and then deactivating the protease by heating, to solve the problem as mentioned above.
Japanese Laid-Open Patent Publication No. 2003-93086 suggests that proteins, peptides, and enzyme can be a cause of allergy. The document also teaches that the method of Japanese Laid-Open Patent Publication No. 63-233750 is clearly problematic in terms of safety in that peptides, which are breakdown products, and proteases still remain, and they have a risk of inducing an allergy. Japanese Laid-Open Patent Publication No. 2003-93086 discloses treating a starting material that contains phospholipid with a phospholipase and then a protease, and then removing any proteins, peptides, and enzymes, to solve the problem as mentioned above. As the procedure of removing proteins or the like, filtration using a filter aid, processing using an adsorbent, and so like are exemplified. It is mentioned that the used adsorbent can be removed by filtration using the filter aid. Although this method is capable of removing proteins and the like, it requires processing such as filtration and adsorption after the reaction, and thus has the problem in that the process becomes more complicated.
Japanese Laid-Open Patent Publication No. 2002-193982 discloses adding a polar organic solvent to an liquid enzyme reaction mixture and then removing hydrophilic impurities, proteins, and inorganic salts from a solution of phosphatidylserine in the organic solvent by water extraction. The document teaches that this can reduce the activity of PLD in the solution of phosphatidylserine to below the detectable limit (0.1 IU/g), however, there is no mention regarding the protein level in the solution.